γδ T cell-intrinsic IL-1R promotes survival during Staphylococcus aureus bacteremia

Staphylococcus aureus is a leading cause of bacteremia, further complicated by the emergence of antibiotic-resistant strains such as methicillin-resistant S. aureus (MRSA). A better understanding of host defense mechanisms is needed for the development of host-directed therapies as an alternative approach to antibiotics. The levels of IL-1, IL-17, and TNF-α cytokines in circulation have been associated with predictive outcomes in patients with S. aureus bacteremia. However, their causative role in survival and the cell types involved in these responses during bacteremia is not entirely clear. Using a mouse model of S. aureus bacteremia, we demonstrated that IL-17A/F and TNF-α had no significant impact on survival, whereas IL-1R signaling was critical for survival during S. aureus bacteremia. Furthermore, we identified that T cells, but not neutrophils, monocytes/macrophages, or endothelial cells were the crucial cell type for IL-1R-mediated survival against S. aureus bacteremia. Finally, we determined that the expression of IL-1R on γδ T cell, but not CD4+ or CD8+ T cells was responsible for survival against the S. aureus bacteremia. Taken together, we uncovered a role for IL-1R, but not IL-17A/F and TNF-α in protection against S. aureus bacteremia. Importantly, γδ T cell-intrinsic expression of IL-1R was crucial for survival, but not on other immune cells or endothelial cells. These findings reveal potential cellular and immunological targets for host-directed therapies for improved outcomes against S. aureus bacteremia.


Introduction
Staphylococcus aureus is a leading cause of bacteremia (1), with a mortality rate of 25% due to the emergence of antibiotic-resistant strains such as methicillin-resistant S. aureus (MRSA) (2). Furthermore, all vaccines to date have failed in clinical trials against S. aureus invasive infections (3,4). Thus, a better understanding of host defense mechanisms is needed for the development of host-directed therapies as an alternative approach to antibiotics.
The IL-1, IL-17, and TNF-a cytokines have been implicated in host defense against S. aureus skin and orthopedic infections (5)(6)(7)(8). Moreover, IL-1, IL-17, and TNF-a cytokine levels in circulation have been associated with predictive outcomes in patients with S. aureus bacteremia (4,(9)(10)(11)(12). For instance, elevated IL-1b at the time of patient admission correlated with reduced duration of the S. aureus bacteremia (11). However, whether the IL-1, IL-17, and TNF-a cytokines have a causative role in host survival and the cell types involved in these responses during S. aureus bacteremia is not entirely clear.
In this study, we evaluated the contributions of IL-1a/b, IL-17A/F, and TNF-a to host survival during S. aureus bacteremia using a preclinical mouse model. Furthermore, we identified the specific cell types that promote host survival using mice with specific deletion of IL-1R on T cells, myeloid cells, neutrophils, and endothelial cells.

Bacterial preparation
The community-acquired methicillin-resistant S. aureus (MRSA) USA300 SF8300 strain, a kind gift from Dr. Binh Diep (UCSF), was cultured in tryptic soy broth (TSB) as previously described (13,14). Briefly, SF8300 was streaked onto a tryptic soy agar (TSA) plate (TSB plus 1.5% bacto agar (BD Biosciences)) and grown overnight at 37°C in a bacterial incubator. Two to three single colonies were picked and cultured in TSB at 37°C in a shaking incubator (240 rpm) overnight (18 h), followed by a 1:50 subculture at 37°C for 2 h to obtain mid-logarithmic phase bacteria. The bacteria were pelleted, washed 3 times with sterile PBS, resuspended in sterile freezing medium (10% glycerol in sterile PBS) at a concentration of 1×10 10 CFU/ml and aliquots stored in cryovials at -80°C until needed. The number of CFUs was confirmed with overnight culture on TSA plates.

Study approval
All mouse strains were bred and maintained under the same specific pathogen-free conditions, with air-isolated cages at an American Association for the Accreditation of Laboratory Animal Care (AAALAC)-accredited animal facility at Johns Hopkins University and handled according to procedures described in the Guide for the Care and Use of Laboratory Animals as well as Johns Hopkins University's policies and procedures as outlined in the Johns Hopkins University Animal Care and Use Training Manual. This study was approved by the Johns Hopkins Animal Care and Use Committee (Protocol #: MO21M378).

Tamoxifen-inducible deletion of IL-1R
The inducible deletion of IL-1R on gd T cells was modified from a previously described protocol (17). The TCRd-IL-1R -/mice were treated daily with 100 ml of 1 mg/ml tamoxifen in sunflower oil injected intraperitoneally for 5 consecutive days. The bacteremia infections were performed 10 days after the last tamoxifen injection. Wild-type (WT) mice were subjected to the same tamoxifen regimen when paired with TCRd-IL-1R -/mice. Tamoxifeninducible deletion of IL-1R was confirmed by flow cytometry, which was comparable to the~60% deletion efficiency in gd T cells in TCRd creER mice based on prior reports (18).

Flow cytometry
For flow cytometric analysis, 100 ml of peripheral blood and spleen was collected from tamoxifen-treated WT and TCRd-IL-1R -/-mice 3h after intravenous infection. Red blood cells were lysed with ACK lysis buffer (ThermoFisher Scientific) and cells were resuspended in FACS buffer (PBS containing 1% BSA and 2mM EDTA). Spleen was manually pushed through a cell separation filter (40 µm) and resuspended in FACS buffer. Single cell suspensions were stained for viability (Viobility 405/520 viability kit, Miltenyi Biotec) and TruStain fcX (Biolegend) was used to block Fc receptor binding. Next, blood single cells were surface stained with the following mAbs: PE-Vio770-CD3 (

Ex vivo CFU enumeration
At 3h post infection, mice were euthanized, and the spleen, liver, and kidneys were harvested and ex vivo CFU were isolated as previously described (5,19). The tissue specimens were homogenized (PRO200 Series homogenizer; PRO Scientific) and then serially diluted and cultured overnight on TSA plates at 37°C. Ex vivo CFU from the homogenized tissue were then enumerated from the plates.

Statistical analyses
Survival rates were compared by log rank (Mantel-Cox) test and data from single comparisons analyzed by Student's t test (twotailed), as indicated in the figure legends. All statistical analyses were calculated with Prism software (GraphPad 9.5 Software, La Jolla, California). CFU data are presented as geometric mean ± geometric standard deviation (SD). All other data are presented as mean ± standard error of the mean (SEM) and values of P <0.05 were considered statistically significant.

IL-1R signaling improves survival during S. aureus bacteremia
The levels of IL-1, IL-17, and TNF-a cytokines in circulation have been associated with predictive outcomes in patients with S. aureus bacteremia (4,(9)(10)(11)(12). Therefore, we set out to determine the mechanistic effect of IL-1a/b, TNF-a, and IL-17A/F on survival during S. aureus bacteremia using a preclinical mouse model whereby 4.8-5.8 × 10 7 CFUs of S. aureus USA300 (SF8300) were injected i.v. and survival measured over time (15,16). To determine the role of IL-1R signaling, we first performed our bacteremia model on wild-type (WT) C57BL/6 and IL-1R -/mice and found that IL-1R -/mice had a statistically significant decrease in survival compared to WT mice ( Figure 1A). Since IL-1a and IL-1b signal through the IL-1R (20), we next tested IL-1a -/and IL-1b -/mice and discovered that both IL-1a -/and IL-1b -/mice had a markedly reduced survival compared to WT mice ( Figure 1A). Next, we examined IL-17A/F -/and TNF-a -/mice and found no statistically significant differences compared to WT mice ( Figure 1B). Taken together, our data indicated that IL-1a and IL-1b signaling via IL-1R enhanced survival during S. aureus bacteremia infections.

gd T cell-intrinsic IL-1R signaling promotes survival during S. aureus bacteremia
Since IL-1R signaling was important for survival during S. aureus bacteremia infections, we next elucidated the specific cell types involved in the IL-1R response. Various cell types use IL-1R signaling to drive host defense and inflammation (20), including myeloid cells, T cells, and non-immune cells (21). Thus, we developed mice with specific deletion of IL-1R in T cells (Lck-IL-1R -/-), myeloid cells (LysM-IL-1R -/-), and neutrophils (S100A8-IL-1R -/-). We also used mice with specific deletion of IL-1R in endothelial cells (VE-Cad-IL-1R -/-), since S. aureus interacts with endothelial cells upon bacteremia infections (22). We discovered that only the Lck-IL-1R -/mice had a significant defect in survival compared to WT mice (Figure 2A), suggesting that IL-1R signaling on T cells, but not myeloid cells, neutrophils, or endothelial cells was important for host survival.
We next determined the specific T cell subset required for IL-1R signaling, since CD4+ and gd T cells are reported to be involved in host defense against S. aureus infections (7,(23)(24)(25). To this end, we developed and tested mice with specific deletion of IL-1R in CD4+ T cells (CD4-IL-1R -/-) and tamoxifen-inducible deletion of IL-1R in gd T cells (TCRd-IL-1R -/-). We discovered that CD4-IL-1R -/mice had no difference in survival compared to WT mice ( Figure 2B). Interestingly, there was markedly decreased survival in TCRd-IL-1R -/mice compared to WT mice ( Figure 2C). There was a trend towards increased circulating gd T cells counts in TCRd-IL-1R -/mice compared to WT mice ( Figure 3A). We confirmed tamoxifen-inducible deletion of IL-1R on gd T cells in the TCRd-IL-1R -/mice by flow cytometry ( Figure 3B). Collectively, IL-1R signaling on gd T cells was important for survival during S. aureus bacteremia infections.

gd T cell-intrinsic IL-1R signaling increases monocyte recruitment to the spleen during S. aureus bacteremia
We next elucidated whether gd T cell-intrinsic IL-1R signaling affected immune cell levels and S. aureus burden during the bacteremia. To this end, we first measured neutrophil, monocyte, and macrophage population levels in the spleens of TCRd-IL-1R -/and WT mice 3 hours post-infection. We found that monocytes, but not neutrophils or macrophages, were significantly decreased in TCRd-IL-1R -/mice compared to WT mice ( Figures 3C-E). Next, we measured S. aureus CFUs in the spleen, liver, and kidney, but found no difference in bacterial burden between TCRd-IL-1R -/and WT mice (Figures 3F-H). These data indicated that gd T cellintrinsic IL-1R signaling promoted monocyte recruitment to the spleen during S. aureus bacteremia.

Discussion
The IL-1, IL-17, and TNF-a cytokines contribute to host defense against S. aureus skin and orthopedic infections (5-8).
Although IL-1, IL-17, and TNF-a cytokine levels in circulation have been associated with predictive outcomes in patients with S. aureus bacteremia (4,(9)(10)(11)(12), whether these cytokines mechanistically promote host survival and the cell types involved in these responses is under-investigated. Thus, we tested mice deficient in IL-1, IL-17, and TNF-a cytokines in a preclinical mouse model of S. aureus bacteremia and discovered that IL-1R signaling was important for host survival. Furthermore, we identified gd T cells as the cell type that drives IL-1R-mediated host survival against S. aureus bacteremia. These results provide several important insights into the protective host responses during S. aureus bacteremia.
First, we found that IL-1R signaling contributed to host survival during S. aureus bacteremia, which aligns with previously published reports (26,27). Similarly, IL-1R signaling promotes host defense against S. aureus skin, orthopedic, and pneumonia infections (5,8,28). Interestingly, we found that both IL-1a and IL-1b were important in our model, suggesting they have non-redundant roles in host survival. This may be explained by the differences in expression profiles between the cytokines. For instance, IL-1a is constitutively expressed in non-immune cell types (29), whereas IL-1b is induced (30). Moreover, IL-1a has a nuclear localization sequence that is absent in IL-1b (31), which has important implications in inflammation (32). Understanding the differential mechanisms of protection between IL-1a and IL-1b against S. aureus bacteremia will be the focus of future work. We also discovered that TNF-a and IL-17A/F did not influence host survival during S. aureus bacteremia at the dose tested. This was unexpected, as both TNF-a and IL-17A/F drive host defense against S. aureus at other infection sites (e.g., skin and orthopedic implants) (5-7, 33, 34). However, in a baboon model of group A streptococcal bacteremia infection, anti-TNF-a monoclonal antibody therapy improved survival outcomes (35). Similarly, heightened TNF-a production correlated with persistent rather than resolving bacteremia in patients (12). Another possibility for the lack of phenotype in TNF-a deficient mice is that lymphotoxin- a signals through the TNF-a receptors (36,37), which may have compensated for TNF-a deficiency in our S. aureus bacteremia model. Although IL-17A did not improve survival outcomes during bacteremia in our model, IL-17A limits the systemic dissemination of S. aureus from skin infection to kidneys (38). Thus, IL-17A may be more important in the control of S. aureus infections in the tissue rather than protection once bacteremia has occurred. Although not analyzed in this study, there may be a role for IL-10 in the infectious process during S. aureus bacteremia, as this cytokine correlates with mortality in humans (11, 12). Collectively, our findings do not support a role for TNF-a and IL-17A/F in survival outcomes during S. aureus bacteremia in our preclinical mouse model. We uncovered that gd T cell-intrinsic IL-1R signaling was crucial for host survival during S. aureus bacteremia. Our findings may relate to prior studies on the protective role of gd T cells and other T cells against S. aureus skin infections and nasal colonization (7,8,(39)(40)(41). In contrast, IL-1R signaling on non-hematopoietic cells was critical for protection against S. aureus skin infections (8). Thus, these findings indicate that the protective cell type that provides the IL-1R signal against S. aureus infections is contextdependent. Given that IL-1R deficient mice succumbed to S. aureus bacteremia within 2 days, our findings suggested that gd T cellmediated IL-1R signaling occurs soon after infection. In fact, gd T cells are an innate source of pro-inflammatory responses driven by IL-1 cytokines independent of T cell receptor engagement (42, 43), perhaps explaining the importance of IL-1R signaling on this T cell subset for rapid protection against S. aureus bacteremia infections. However, since IL-17A/F cytokines were not important for host survival herein, and gd T cells produce IL-17 cytokines in response to IL-1R signaling (42), it begs the question of how gd T cellintrinsic IL-1R signaling is mediating protection against S. aureus bacteremia? Our findings suggested that gd T cell-intrinsic IL-1R signaling promotes monocyte recruitment to the spleen during S. aureus bacteremia as a mechanism of protection. This may relate to the known role of IL-1b to induce the monocyte-recruiting chemokine, CCL2 (44,45). Other potential explanations include gd T cell production of antimicrobial peptides, IL-22, and neutrophil recruiting chemokines to promote host survival (46,47), which have been associated with protection against S. aureus at other infection sites (13,48). Understanding the localization and mechanism of protection of the gd T cell-specific IL-1R response during S. aureus bacteremia will be part of our future interrogations.
There were limitations. For instance, our study was conducted with a single S. aureus strain, limiting the broader conclusions of our findings. However, other studies have tested additional S. aureus strains in IL-1R deficient mice or mice treated with IL-1Ra with similar results (26,27,49), suggesting that IL-1R-mediated survival is not specific to a single S. aureus strain. Moreover, we used a high inoculum of S. aureus in the bacteremia model (i.e., LD90), which may have missed phenotypes present in a lower inoculum (e.g., LD50). Another limitation to the study is the possibility that the phenotypes in our cell-specific IL-1R deficient mice are due to changes in cytokine production in IL-1a and IL-1b rather than IL-1R-specific mechanisms. The use of tamoxifen to delete IL-1R in the TCRd-IL-1R -/mice may have influenced the immune responses upon the S. aureus bacteremia infection (e.g., neutrophil function) (50), which was observed in Figure 2C. To control for these effects, we similarly treated the control WT comparison group with tamoxifen. Importantly, deletion efficiency in gd T cells in Lck cre and TCRd creER mice is ∼20% and ∼60%, respectively (18,51), leaving the possibility that IL-1R signaling on other T cell subsets not specifically tested in this study (e.g., NK T cells and MAIT cells) contributed to host survival during S. aureus bacteremia infections. Addressing these limitations will be performed in our future work.
Taken together, the results of this study indicate that gd T cellintrinsic IL-1R signaling promotes host survival during S. aureus bacteremia infections. Thus, IL-1R on gd T cells may serve as a hostdirected therapeutic target for the treatment of S. aureus bacteremia infections and potentially other antibiotic-resistant infections. Potential therapeutic strategies could include IL-1R agonism or neutralizing the IL-1R antagonist (IL-1Ra) to promote survival during S. aureus bacteremia. However, further studies are warranted to understand the protective mechanisms of gd T cellintrinsic IL-1R signaling against S. aureus bacteremia.

Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement
The animal study was reviewed and approved by the Johns Hopkins Animal Care and Use Committee (Protocol #: MO21M378).

Author contributions
YW and NA conceived and designed the study. YW conducted the experiments. YW, MA, DD, and CY collected the data. YW analyzed the data. YW and NA wrote the manuscript. All authors reviewed the final version of the manuscript.

Funding
This study was funded in part by grants R01AR073665 (NA), R01AI111205 (NA), and R01AI146177 (NA) and from the United States National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) and National Institute of Allergy and Infectious Diseases (NIAID).

Conflict of interest
NA has received previous grant support from Pfizer and Boehringer Ingelheim and has been a paid consultant for Janssen Pharmaceuticals.
The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.
Publisher's note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.